Development of new therapies against melanoma, a nearly incurable disease if allowed to progress to the deadly metastatic stage, is a well-recognized medical need. As in all other cancers, tumorigenesis and tumor progression in melanoma is accompanied by substantial rewiring of central carbon metabolism opening unexplored opportunities for diagnostics and treatment. Recent studies of the investigators of this application implicated a substantially enhanced utilization of glutamine as an essential anaplerotic source of carbon and energy and a potential therapeutic target in human melanoma cell lines. The preliminary results of these studies led to the specific hypothesis about the critial importance of the cataplerotic exit of glutamine- originating carbon units from the tricarboxylic acid cycle, which may provide unique targeting opportunities. The proposed studies will address this hypothesis using an established integrative approach, which combines multifaceted metabolic analyses, including stable-isotope tracing, with genetic suppression of selected target enzymes over a panel of representative human melanoma lines followed by testing and validation of main finding in mouse xenograft models. In addition to gaining fundamental mechanistic insights, anticipated deliverables of the project would include validated biomarkers (signature metabolites and/or fluxes) and potential target enzymes. The proposed research plan includes two major specific aims featuring in vitro and in vivo studies: (I) to elucidate mechanisms and identify potential diagnostic and therapeutic targets involved in the key aspects of glutamine metabolism in a range of melanoma cell lines by a synergistic combination of techniques including targeted metabolomics, physiology and cell biology; (II) to validate the key aspects of glutamine metabolism, biomarkers and tentatively identified targets in the xenograft mouse models of primary melanoma tumors and metastases. If successfully accomplished, this project would provide a strong starting point for advanced translational studies and therapeutic developments